Methods of donor specific crossmatching

ABSTRACT

The detection of endothelial cell antibodies has been proven clinically important for successful organ transplantation. Disclosed are methods of isolating endothelial cell antibodies for donor-specific crossmatching.

RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 10/439,666, filed May 16, 2003 which claims the benefit of U.S. Ser. No. 60/381,033, filed May 16, 2002, the content of which is incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to a method for the direct isolation of endothelial cells from whole blood for routine donor-specific crossmatching to detect anti-endothelial cell antibodies prior to organ transplantation.

BACKGROUND OF THE INVENTION

The presence of donor lymphocyte-reactive Human Leukocyte Antigen (HLA)-specific antibodies either before and/or after renal allograft transplantation has been associated with hyperacute rejections, early acute rejections, and poor graft survival. However, rejections may occur in the absence of detectable lymphocytotoxic antibodies, suggesting that non-HLA antigenic systems may also play a role in renal allograft hyperacute and acute rejections. Antibodies reactive with endothelial cells and monocytes (also called the EM-antigenic system), or only with endothelial cells, have been described and reported to have a deleterious effect in several organ transplantations.

Recently, the major histocompatibility class I-related chain A antigen (MICA) expressed on endothelial cells was identified as one of the target antigens of humoral immunity associated with irreversible rejections of kidney allografts. Studies of HLA-identical living-related donor allografts showed that the presence of endothelial cell/monocyte reactive antibodies correlated with rejection, graft loss, and poor allograft function. It was reported that this reactivity could be responsible for up to 80% of irreversible rejections in this group of patients. However, the routinely used lymphocyte cross-match (LXM) does not permit detection of the clinically relevant HLA class-I, class-II, endothelial/monocyte-reactive and endothelial cell-specific antibodies. Although the presence of circulating endothelial cells in whole blood has been a subject of debate for many years, the existence of circulating precursor endothelial cells in adult humans has recently been reported by some investigators. However, there is currently no suitable method available to perform a routine donor-specific endothelial cell-crossmatch (ECXM).

Therefore, there is a need to efficiently perform routine donor-specific endothelial cell cross-matching to aid in the identification of better donor-recipient combinations, which will thereby have a greater impact on transplant survival than the current method of lymphocyte cross-match.

SUMMARY OF THE INVENTION

The invention pertains to a method of isolating endothelial cells which are useful in donor specific crossmatching prior to transplantation. The isolated endothelial cells are also useful in diagnosing various vascular and immune related disorders.

The invention includes a method of isolating an endothelial cell from a biological sample by providing a sample known to contain, or suspected of containing, an endothelial cell. The endothelial cell is Tie-2 positive. Alternatively, the isolated endothelial cell is VEGFR positive. The isolated endothelial cell is a mature cell. Alternatively, the isolated endothelial cell is an endothelial cell precursor. The sample is contacted with a detection reagent to form a detection reagent-endothelial cell complex. The endothelial cell is isolated by separating the complex form the sample. Separation is accomplished, for example, by flow cytometry or use of a magnetic field.

The detection reagent is a ligand for an endothelial sell surface receptor such as angiopoietin or vascular endothelial growth factor (VEGF). Alternatively the detection reagent is an antibody specific for an endothelial cell surface receptor such as Tie-2 or the vascular endothelial growth factor receptor (VEGFR). The antibody is an intact antibody or an antibody fragment (e.g. Fab, or Fv). The detection reagent is attached to a solid support, such as a non-magnetic, magnetic, or paramagnetic bead.

The biological sample is whole blood, sera, tissue homogenate, peripheral blood mononuclear cells (PBMCs) or leucapherisate.

The invention further provides a method of cross-matching a donor and a recipient by providing a biological sample from a donor that is known to contain an endothelial cell, contacting the donor sample with detection reagent to isolate an endothelial cell. A recipient biological sample is screened for reactivity with the donor isolated endothelial cell. No reactivity between the recipient sample and the isolated endothelial cell indicates compatibility between the donor and recipient and a higher probability that the organ transplant will be successful.

In a further aspect, the invention includes a method of diagnosing an immune related or vascular disorder in a subject by providing a first sample from a subject known to contain, or suspected of containing, an endothelial cell. The first sample is contacted with a second sample. The second sample is from a subject known to contain, or suspected of containing, an auto antibody. Alternatively the second sample contains reagents (e.g. antibodies) that recognize endothelial cell surface markers associated with the particular disorder. Complex formation between the endothelial cell and the second sample is determined. The presence of the complex indicates the disorder in the subject.

In yet another aspect, the invention includes a method of determining the efficacy of treatment or prognosis of an immune related or vascular disorder in a subject by providing a first sample from a subject known to contain, or suspected of containing, an endothelial cell, contacting the endothelial cell with a second sample from a subject known to contain, or suspected of containing, an autoantibody, measuring any autoantibody-endothelial cell precursor complex present to yield a subject profile. The subject profile is compared with a reference profile, wherein a similarity between the subject profile and reference profile indicates that the treatment is efficacious or a favorable prognosis.

Immune related or vascular disorders include vasculitis, atherosclerosis, bleeding disorders, angiogenesis, thrombosis, defective wound healing, and transplantation.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dot plots representing forward and side scatter profile as well as fluorescence of peripheral blood mononuclear cells (PBMCs) stained with anti-Tie-2+ monoclonal antibodies (Mabs). Tie-2+ cells (grey dots) appeared in the lymphocyte gate. Histograms show the percentage of Tie-2+ cells (app. 2±3%) in PBMCs stained with negative control abs and Tie-2 Mabs.

FIGS. 2 a-c are photographs of the morphology of Tie-2+ cells under light microscope at various time points after isolation from peripheral blood. Initially, these cells appeared either as single cells or as clusters of round cells (a), which after few days of culture converted into adherent cells with extended cytoplasm, (the dark objects are beads) (b). After 7-12 days in culture the cells gradually developed a spindle shape (c).

FIG. 3 a is a series of photographs showing Tie-2+ cells stained positive for the endothelial associated markers, acetylated low density lipoprotein (Ac-LDL), von Willebrand factor (vWF), vascular endothelial growth factor receptor-1 (VEGFR-1) and the vascular cell adhesion molecule (VCAM) (on activated Tie-2+ cells). Tie-2+ cells also constitutively expressed the human leukocyte antigen class I (HLA class I) and the monocyte/macrophage marker CD68.

FIG. 3 b shows that quiescent Tie-2+ cells expressed small amounts of the clinically important MICA antigens (dark grey), and HLA class II (light grey), control antibodies (black).

FIG. 4 a is a dot plot that represents the forward and side scatter of rosettes of Tie-2+ cells and paramagnetic beads (gate R1) and paramagnetic beads only (gate R2).

FIGS. 4 b-g are histograms showing that pre-transplant sera from two kidney-transplanted patients with hyperacute rejections reacted strongly with their respective donor-specific Tie-2+ cells (b & e), while their Tie-2− fractions, which included lymphocytes did not (d & g). Histograms c & f show that the paramagnetic beads alone did not react with the sera non-specifically. The grey lines represent control or negative sera, and the black lines represent reactivity with patient's sera.

FIG. 5 is a series dot plots representing forward and side scatter profile as well as histograms representing fluorescence of endothelial precursor cells isolated from blood using para magnetic particles with a diameter of 100-350 nm coupled to antibodies specific for Tie-2. For fluorescence the endothelial precursor cells have been stained with antibodies against various surface molecules.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based in part on the discovery that targeting a single specific cell population allows for the detection of clinically relevant donor-specific human leukocyte antigens (HLA) class I, class II, endothelial-monocytes, or endothelial cell-specific antibodies prior to transplantation of organs. Routine use of the endothelial cell cross matching will aid in identifying better donor-recipient combinations and thus have a greater impact on transplant survival as compared to traditional lymphocyte cross matching.

The clinical importance of endothelial cell (EC) antibodies in allo-transplantation has been reported. However, lack of a suitable method for isolation of donor-specific ECs has prevented routine detection of these antibodies prior to transplantation. The invention provides a quick and simple method for the direct isolation of ECs from whole blood, for routine cross-matching to detect anti-EC antibodies. The presence of endothelial cell-reactive antibodies has previously been detected using human umbilical vein endothelial cell (HUVEC) lines, keratinocyte cell lines, or monocytes as targets, or by immunohistochemistry. However, these methods are cumbersome because endothelial cell culturing is tedious and a generally large panel of donors of HUVEC lines/keratinocytes is required to represent all the known polymorphic alleles for screening of endothelial cell-reactive antibodies. Moreover, these methods do not permit the detection of donor-specific endothelial cell antibodies. Thus, use of an endothelial cell cross-match is advantageous over use of lymphocyte cross-match because lymphocyte cross-match does not allow for such detection or isolation of donor-specific endothelial cell-reactive antibodies.

ECs were isolated using magnetic beads coated with antibodies against the angiopoietin receptor, Tie-2 that is expressed on EC precursors. Tie genes play an important role in renal vascular development, and based on transplantation experiments these precursors have been shown to contribute to the generation of glomerular maturation. A retrospective analysis of 50 previously well characterized crossmatch sera taken immediately prior to transplantation from patients with end-stage kidney disease were tested. Tie-2+ cells expressed HLA class I, class II and other endothelial cell markers. Sera known to contain only EC specific or EC and monocyte (EM) reactive antibodies reacted positively with Tie-2+ cells, but not with Tie-2− cells from the same individual. In addition, the Tie-2+ cells reacted with sera containing only HLA class I or class II abs. In all, 3/25 sera from patients with stable graft outcome and no rejections reacted with Tie-2+ cells. This antigen-antibody interaction is relevant to the pathogenesis of rejection, since in many studies these antibodies are not detected in the serum of patients with good graft function or in non-transplantation patients.

Methods of Isolating Endothelial Cells

The invention includes methods of isolating an endothelial cell from a mixture of cells by contacting the mixture of cells with a detection reagent to form a endothelial cell-detection reagent complex. The complex is formed via a specific affinity interaction between the detection reagent and the cell. The complex is separated from the mixture to isolate the endothelial cell. The complex is separated from the mixture using techniques known in the art, such as by, e.g., liquid chromatography (e.g., HPLC or FPLC), High Performance Membrane Chromatography (HPMC), flow cytometry or use of a magnetic field. Alternatively, the complex is separated from the mixture by attaching the detection reagent to a solid support. A washing step may be employed by re-suspending the complex in a biologically compatible solution. The complex can be re-suspended, i.e., washed as many times as desired. Typically the particles are washed three times. A biologically compatible solution include biological buffers known in the art such as phosphate buffer saline (PBS).

The detection reagent is an endothelial cell detection reagent. An endothelial cell detection reagent is any reagent that specifically identifies an endothelial cell. For example, the detection reagent is a ligand for a endothelial cell surface receptor. The endothelial cell surface receptors include, for example, the EC-specific tyrosine kinase receptor Tie-2 or VEGFR. The ligand is for example angiopoietin, VEGF or an antibody specific for the cell surface receptor. The antibody is a monoclonal antibody or a polyclonal antibody. The term antibody encompasses not only an intact antibody, but also an immunologically-active antibody fragment, e.g. , a Fab or (Fab)₂ fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin. For example the detection reagent is monoclonal antibody Tie-2 or VEGFR-1

The detection reagent is attached to a solid support. The solid support is a particle, a polymer (e.g., polystyrene, polyethylene), a vessel, a chamber, a dipstick, beads, particles, membranes (e.g., nylon, nitrocellulose or polyvinylidenedifluoride (PVDF)), or other forms known in the art.

The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups. The solid support may be positively charged, negatively charged or hydrophobic. Functionalized coated supports for use in the present invention may be prepared by modification of the support. For example, uncoated support is treated with a polymer carrying one or such functional groups, such as polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups, or an aminoalkylated polymer to provide amino groups. U.S. Pat. No. 4,654,267 describes the introduction of many surface coatings.

The particle is made of metal compounds, silica, latex, polymeric material, or a silica, latex or polymer nuclei coated with a metal or metal compound. Preferably, the particle is made of a metal compound, such as iron, gadolinium, zinc, indium, gold, silver, cobalt, copper, or magnesium. Most preferably, the particle is magnetizable or magnetic. By “magnetizable or magnetic” is meant that the particle is capable of having a magnetic moment impaired to it when it is placed in a magnetic field.

The detection reagent is labeled with a detectable marker. For example the detection reagent to labeled with a radioactive isotopes (e.g., ¹²⁵I, and ¹³¹I) , enzymes (e.g., peroxidase, beta.-galactosidase, alkaline phosphatase) or fluorescent substances (e.g., fluorescein isothiocyanate (FITC). The labels are quantified by the conventional methods well-known in the art, thereby the formed immune complex is quantified. The mixture of cells is any sample known to or suspected of containing an endothelial cell. For example the mixture is a biological sample such as whole blood, sera, leucapherisate, bone marrow, peripheral blood mononuclear cells or a tissue homogenate.

An endothelial cell is any cell derived from any part of the vascular tree. For example the endothelial cell is from large and small veins and arteries capillaries, the umbilical vein of newborns, blood vessels in the brain or from vascularized solid tumors. The endothelial cell is a mature cell. Alternatively, the endothelial cell endothelial cell precursor. Preferably, the endothelial cell is Tie-2 positive.

Methods of Donor Specific Crossmatching

Cross-matching detects those antigenic differences to which the recipient is already sensitized. A donor is crossmatched to a recipient by contacting a donor sample with a detection reagent to isolate an endothelial cell. The recipient sample is contacted with the isolated endothelial cell and reactivity of the recipient sample with the isolated endothelial cell is determined. By “reactivity” it is meant that that a complex is formed via a specific affinity interaction between the recipient sample and the cell. No reactivity of the recipient sample with the isolated endothelial cell indicates compatibility between the donor and recipient sample. In contrast, reactivity of the of the recipient sample with the isolated endothelial cell indicates non-compatibility between the donor and recipient sample. Compatibility is measured by no or low hyperacute rejection of the donor transplant by the recipient.

The donor and the recipient are, e.g., any mammal, e.g., a human, a pig, a cow, or a horse. The donor and the recipient are the same species. Alternatively, the donor and the recipient are of different species.

The donor and recipient sample is, for example, whole blood, sera, leucapherisate, bone marrow, peripheral blood mononuclear cells or a tissue homogenate. Optionally, the samples are subjected to a pre-purification step prior to crossmatching.

Reactivity is determined my methods known in the art. For example reactivity is measured by an ELISA assay, flow cytometry (e.g. flow cytometric cross-match), or complement-dependent lymphotoxicity cross-match.

Vascular and Immune Disorders

The invention also provides methods of diagnosing, accessing the prognosis or monitoring the course of treatment of vascular and immune disorders.

In these methods a first test sample is provided from a subject The sample is known to or suspected of containing an endothelial cell. Optionally, the endothelial cell is isolated from the first sample.

An immune disorder is diagnosed by contacting the first sample to a second sample from the subject known to contain, or suspected of containing, an auto-antibody and identifying an autoantibody-endothelial cell complex. The presence of an autoantibody-endothelial cell complex indicates the subject is suffering from or predisposed to an immune disorder. In contrast, the absence of an autoantibody- endothelial cell complex indicates the subject is not suffering from or predisposed to an immune disorder.

A vascular disorder is diagnosed by contacting the first sample to a second sample. The second sample is derived from the subject. Alternatively, the second sample comprises antibodies to cell surface markers know to be associated with a particular vascular disorder The presence of a second sample-endothelial cell complex indicates the subject is suffering from or predisposed to a vascular disorder. In contrast, the absence of a second sample-endothelial cell complex indicates the subject is not suffering from or predisposed to a vascular disorder.

The methods allow the course of treatment of a vascular or immune to be monitored or the prognosis of the subject to be determined. In this method, a test sample is provided from a subject undergoing treatment for the disorder. If desired, test samples are obtained from the subject at various time points before, during, or after treatment. The presence of an endothelial cell complex is then determined to create a subject profile. The subject profile is compared to a reference profile whose vascular disorder or immune disorder state is known. The reference profile has not been exposed to the treatment. The reference profile is derived from a sample type as similar to test sample. Optionally, the reference profile is derived from a database of molecular information derived from samples for which the assayed parameter or condition is known.

If the reference profile contains no autoantibody-endothelial cell complexes, a similarity in the amount of complexes between the subject profile and the reference profile indicates that the treatment is efficacious (e.g., that one or more symptoms of the immune disorder are alleviated or that the severity of the disorder is reduced), and thus, a favorable prognosis for the subject. However, a shift in the amount of complexes between the subject profile and the reference profile indicates that the treatment is not efficacious, and thus, an unfavorable prognosis for the subject.

When the reference profile contains autoantibody-endothelial cell complexes, e.g., when the reference profile includes complexes taken from the subject at the time of diagnosis but prior to beginning treatment, a similarity in the expression of complexes pattern between the subject profile and the reference profile indicates the treatment is not efficacious. In contrast, a shift in expression complexes in the subject profile and this reference profile indicates the treatment is efficacious.

By “efficacious” it is meant that the treatment leads to a decrease in any of the symptoms of an autoimmune disorder in a subject previously noted. When treatment is applied prophylactically, “efficacious” means that the treatment retards or prevents an immune related or vascular disorder.

An immune disorder includes disorders mediated by an immune mechanism such as deposition of immune complexes, inflammation, direct attack by circulating antibodies (e.g., autoimmune disorders). An autoimmune disorder or an autoimmune related disorder includes those disorders caused by an immune response against the body's own tissues. Autoimmune disorders result in destruction of one or more types of body tissues, abnormal growth of an organ, or changes in organ function. The disorder may affect only one organ or tissue type or may affect multiple organs and tissues. Organs and tissues commonly affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissues, endocrine glands such as the thyroid or pancreas, muscles, joints, and skin. Autoimmune disorders include for example, autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease, chronic fatigue syndrome, Crohn's disease, Hashimoto's thyroiditis, fibromyalgia, systemic lupus erthyematosus, Graves' disease, idiopathic thrombocytopenia purpura, multiple sclerosis, psoriasis, rheumatic fever, rheumatoid arthritis.

Vascular disorders include disease associated with the vascular system. For example vasculitis, atherosclerosis, bleeding disorders, defective wound healing.

Symptoms of an autoimmune disorder depend on the specific disease and the organ or tissue that is affected. For example, systemic lupus erythematosus may cause kidney failure, arthritis, and a skin rash on the face. Autoimmune hemolytic anemia causes anemia, or low red blood cell counts. Generally symptoms of autoimmune disorders may include: low-grade fever, malaise, which is a vague feeling of illness, fatigue, or tiring easily. Autoimmune disorders are diagnosed based on symptoms, a physical exam, and the results of blood tests.

Treatments to reduce symptoms may include: nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin or ibuprofen, to relieve fever, joint pain, and muscle aches corticosteroids, or steroids, help reduce inflammation. These medications are often used on a short-term basis to get a person through a sudden episode or flare-up, medications to suppress the immune system, such as methotrexate, azathioprine, and cyclophosphamide, which help to reduce inflammation and organ damage. In some cases, other treatments may be needed. For example, surgery may be needed for blockage of the bowels, which may occur in Crohn's disease. Blood transfusions may be needed in severe cases of autoimmune hemolytic anemia. Insulin is given to individuals with type 1 diabetes to control blood glucose levels.

The subject is preferably a mammal. The mammal can be, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.

Kits

The invention further provides kit for the isolation of an endothelial cell precursor cell fro a sample according to the methods of the invention. The kit contains one or more antibodies (i.e., first antibody that specifically binds an endothelial cell precursor cell surface marker, and a detection reagent. Endothelial precursor cell surface markers include for example, Tie2, HLA II, CD31, CD105, CD151, VEGFR-2, CD133, VEGFR-1, CD68, CD14, CD62E, CD106, CD54, VWF or Ac-LDL. The sample is any sample known to or suspected of containing an endothelia cell precursor cell. For example, the sample is blood or a component thereof, e.g. PBMCs.

The detection reagent a compound that specifically binds human immunoglobin, e.g. IgG or IgM. For example the detection reagent is example an antibody (i.e., second antibody.

Optionally, the kits contains a positive or negative control or both, instruction for using the kit (e.g., written, tape, VCR, CD-ROM, etc.), sample collection means. The collection means contains an anti-coagulant such as an anti-coagulant heparin. Sample collection means are well known to those skilled in the art. For example, the sample collection means is a CPT vacutainer tube.

A positive control is for example a polypeptide that contains an antigenic determinant of an endothelial cell precursor cell surface marker protein, where the antigenic determinant binds to antibody. Whereas a negative control is a polypeptide that does not contain an antigenic determinant of an endothelial cell precursor cell surface marker protein

The antibody is a monoclonal antibody. Alternatively, the antibody is a polyclonal antibody. Optionally, the antibody is a recombinant antibody. The antibdody is an antibody fragment such as F_(ab), F_(ab)′ and F_((ab′)2) fragments. By “specifically bind” or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity (K_(d)>10⁻⁶) with other polypeptides. The reagents are packaged in separate containers, e.g., an antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix), a control reagent (positive and/or negative), and/or a detection reagent.

The assay format is any immunological assay format known in the art. For example, the assay format is an ELISA, particle assay or strip assay.

For example, first antibody, is immobilized on a solid matrix such as a particle microtiter plate, a cuvette, a tube, or a strip. Particles include microparticles such as paramagnetic particles.

GENERAL METHODS

The data described herein was generated using the following reagents and methods.

EXAMPLE 1 Coupling of Anti-Tie-2 Mabs to Magnetic Beads

Mouse anti-human Tie-2 monoclonal abs. (Mabs) (BD Pharmingen, Oxford, UK) were first coupled to pan-mouse Dynabeads with a DNA-linker (cat. no. 115.19) (DYNAL, Oslo, Norway). For this purpose, 10 μg of Tie-2 Mabs were added to 500 μls of pan-mouse Dynabeads. The bead-Mab suspension was rotated on a rock n' roller for 24 hrs at 4° C. Excess Mab was removed and the Tie-2 Mab coated beads were blocked with 2 ml phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) on a rock n' roller at 4° C. for 10 min. The blocking step was repeated six times after which, the beads were resuspended in original volume (500 μs) of PBS/0.1% BSA.

EXAMPLE 2 Isolation of Tie-2+ Cells from Peripheral Blood Mononuclear Cells (PBMC)

Due to the large numbers of cells required to establish the specificity of the Tie-2+ cells, in the initial experiments PBMCs were isolated from leucapherisate of healthy blood donors by gradient centrifugation with lymphoprep (Nycomed-Oslo, Norway). ECs were isolated from PBMCs using anti-Tie-2 Mabs coated magnetic beads. PBMCs were first distributed into several tubes each containing 40×10⁶ cells. Fifteen μls of pre-coated Tie-2 magnetic beads were added to each tube and incubated in a volume of 500 μls RPMI medium (GIBCO, Paisley, UK) supplemented with 2 mM L-glutamine and 10% heat inactivated fetal calf serum. Cells+beads were incubated on rock 'n roller at 4° C. for 30 min. Tie-2+ cells were separated after extensive washing (5-6 times) with PBS using a magnet. Tie-2+ cells from all tubes were pooled together and rosettes were counted under a light microscope.

EXAMPLE 3 Isolation of Tie-2+ Cells Directly from Peripheral Blood

40 ml-heparinized blood was obtained from normal healthy donors. The blood was washed once as follows: 10 ml blood was diluted with 40 ml PBS/0.1% BSA. The blood was centrifuged at 800 g for 10 min. The supernatant was discarded and the blood cells were resuspended in 10 ml of PBS containing 0.6% sodium citrate. Fifty μls of Tie-2 Mabs coated magnetic beads were added to each tube and incubated for 30 min at 4° C. on a rock 'n roller. Tie-2+ cells were collected by a magnet and washed once with PBS-Na-citrate. After 4-5 washes with PBS, Tie-2+ cells were collected and the concentration of the cells adjusted to approximately 3-4×10⁶ cells/ml. These cells surrounded by beads were used either in a microcytotoxicity assay or the flow cytometer.

EXAMPLE 4 Immunocytochemistry

The Mabs used for immunocytochemistry and FACs analysis are given in Table 1. Tie-2+ cells obtained from leucapherisate of normal volunteers gave sufficient numbers of cells to perform various immunocytochemical analysis. Tie-2+ cells were grown on fibronectin-coated tissue culture plates. Cells were allowed to attach (24 hrs) prior to staining for various EC-specific markers. Cells were left either untreated or stimulated with TNF-α and IFN-γ for 14 hrs. For immunocytochemistry, cells were fixed using 30% acetone in methanol for 1 min. After two washes with PBS, cells were blocked using 1% bovine serum albumin (BSA) in PBS for 1 hr at room temperature. The cells were washed twice with PBS and incubated with the above mentioned primary antibodies diluted 1:100 (in PBS) at 4° C. for 2 hrs. The secondary antibody was a goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) diluted 1:500 (in PBS). After incubation for 1 hr at 4° C., the cells were washed twice and analysed under a fluorescence microscope.

EXAMPLE 5 Flow Cytometric Assay for Detection of Anti-Endothelial Cell Antibodies

A total of 50 sera from kidney-transplanted patients were studied. Over the past years (1988-2001) the pre-transplant sera from kidney patients has been meticulously characterized, using various methods, for the presence of endothelial cell specific, endothelial-monocyte reactive, and HLA antibodies found to be associated with rejections. During these years 15 pre-tranplantation sera, characterized as having either anti-EM or EC specific antibodies from non-alloimmunized patients with rejections, have been collected.

The general methods described herein are based on these 15 sera. Previous characterization showed that ten sera gave positive reactions with human umbilical vein endothelial cell (HUVECs) lines and monocytes (Table 2, pt. nos. 1-10), and five sera with only ECs (Table 2, pt. nos. 11-15). Since none of the fifteen patients with EM or EC-specific antibodies had been alloimmunized, no detectable HLA alloantibodies were found in these sera. In addition, ten well-characterized sera from alloimmunized patients known to contain only HLA class I or only class II alloantibodies were selected (Table 2). As controls, 25 sera from patients with no graft rejections were also tested.

Using these sera, it was determined whether the isolated Tie-2+ cells could be suitable targets for detection of clinically relevant antibodies in kidney organ transplantation. A pool of sera from patients who had formed alloantibodies as a result of multiple blood transfusions or organ transplantations was used as a positive control. Sera from healthy non-transfused blood group AB males served as negative controls. The following antibodies, FITC-conjugated F(ab′)₂ fragments of goat anti-human IgG (Fc specific), or IgM (Immunotech, USA) were used. For the flow cytometric ECXM 100,000 Tie-2+ cells coupled to beads were used and the procedure carried out as described earlier. The cells were analyzed on a Becton Dickinson flow cytometer (FACSorter). A shift in the mean fluorescence of 10 channels in the test sample as compared to negative control was considered as positive, determined as described before. This value is arbitrary and should be determined by each transplant laboratory. The Tie-2+ cells were also used in the routinely performed immuno-magnetic microcytotoxicity assay as described elsewhere. In addition, Tie-2+ cells immediately after isolation were tested in the flow cytometer for all the endothelial cell surface markers (Table 1).

EXAMPLE 6 Immunocytochemistry and FACS Analysis of Endothelial Cell Markers on Isolated Tie-2+ Cells

Tie-2+ ECs were isolated from human peripheral blood by magnetic bead selection. FACS analysis showed that approximately 3±4% of PBMCs were Tie-2+ (FIG. 1). After 24 hours in culture, the majority of Tie-2+ cells attached to fibronectin-coated 24 well plates and became adherent. Initially, these cells appeared either as single cells or as clusters of round cells, which after few days of culture converted into adherent cells with extended cytoplasm. After 4 days in culture the cells gradually developed spindle shape (FIG. 2 a-c).

FIG. 3 and Table 1 summarize the results of the immunocytochemical analyses using antibodies to known antigens of endothelial cells. Already at 0 hours, FACS analysis indicated that Tie-2+ cells expressed the acLDL-receptor, vWF, VEGFR-1, and strong expression of HLA class I and HLA class II antigens. Importantly, the cells expressed the clinically important MICA antigen, though weakly. Upon cytokine activation, the cells expressed both the endothelial cell specific adhesion molecules CD62 E (E-selectin) and CD 106 (VCAM), as well as increased expression of HLA class II. Thus, both the immunocytochemical and FACS analysis indicated that Tie-2+ cells expressed majority of the endothelial cell markers.

EXAMPLE 7 Detection of Anti-Endothelial Cell Reactive Antibodies

On an average, approximately, 3±4×10⁴ Tie-2+ cells/10⁶ PBMCs can be obtained from single donors. Since a large number of sera were analyzed herein Tie-2+ cells from leucapherisate of healthy blood donors were isolated. The results are shown in Table 2. All the 10 sera known to have endothelial-monocyte antibodies and 5 sera with endothelial cell-specific antibodies showed varying patterns of reactivity with the panel of Tie-2+ cells from six different donors. However, the Tie-2− fractions, which included the lymphocytes from the same donors, did not react with any of the sera. The reactivity pattern with the Tie-2+ cell panel of both the endothelial-monocyte and endothelial cell-specific antibodies as seen in Table 2, indicates the existence of polymorphism in these antigenic systems or alternatively presence of antibodies with varied specificities.

Sera known to contain only HLA class I broadly reactive alloantibodies also gave positive reactions in every instance with Tie-2+ cells from single donors. Reactivity of HLA class II alloantibodies (limited specificities) was also observed with unstimulated Tie-2+ cells. 3/25 (12%) sera from control patients with no rejections and with stable graft functions gave positive reactions with four of the Tie-2 panel donors (Table 2). Results obtained using the immuno-magnetic microcytotoxicity assay were in agreement with the flow cytometric analysis (Table 3).

EXAMPLE 8 Donor-Specific Endothelial Cell Cross-Match

Donor-specific endothial cell crossmatches were retrospectively performed in two cases where frozen donor peripheral blood mononuclear cells were available. The first kidney grafts of both patients were lost in hyperacute rejections in the absence of demonstrable donor-specific HLA antibodies. Cross-match sera from one of these patients had been previously well characterized as having anti-endothelial cell-specific antibodies using HUVECs.

In this instance, Tie-2+ cells from the peripheral blood mononuclear cells of this patient's (point. no. 11, Table 2) first donor (father) were isolated and kept frozen in liquid N₂. A cross-match with the serum of the patient taken immediately prior to the first transplant was retrospectively performed. The results are shown in FIG. 4 b-d. The results from a second case (point. no. 12, Table 2) are very similar to point no. 11, and are shown in FIG. 4 e-g. As seen, in Table 2, the sera from both these points gave similar pattern of reactivity with Tie-2+ cell panel. No reactivity with the Tie-2− fractions were observed.

EXAMPLE 9 Evaluation of Cell Surface Marker Expression of Tie-2 Positive Cells

Cell surface marker expression of endothelial precursor cells isolated using anti-Tie-2 magnetic particles was evaluated.

Blood was collected in standard vacutainer tubes containing an anti-coagulant such as EDTA, ACD or heparin or in CPT vacutainer tubes from BD preclinical solutions. PBMC was separated using a Ficoll gradient through centrifugation at 2000 rpm in 20 minutes without brakes or by spinning the CPT tubes according to instructions from the manufacturer.

Paramagnetic particles with a sixe distribution between 100-350 nm coupled to antibodies specific for Tie-2 were added to the PBMC and incubated on ice for 30 minutes with gentle tilting. After incubation 3 ml of PBS was added to the particle-cell suspension and the tube was placed in a standard magnet. After two minutes the fluid was removed and the separated cells resuspended in PBS. The cells were washed by repeating this procedure.

The separated cells were analyzed using FACS (Fluorescence Activated Cell Sorter) by adding antibodies specific for different surface markers that was conjugated with fluorochromes. Results are shown in FIG. 5 and Table 4.

EXAMPLE 10 Evaluation of Anti-Coagulanst for the Isolation of Endothelial Cell Precursor Cells

Various anti-coagulants were tested for use of separation of PBMC for the isolation of endothelial cell precursor cells. As shown in Table 5, the anti-coagulant heparin is superior to other anti-coagulants such as EDTA, ACD or Citrate as it results in higher yield of endothelial cell precursor cells.

EXAMPLE 11 Instructions for Use XM-ONE™

-   -   1) Collect blood in CPT vacutainer tubes following standard BD         Vacutainer™ procedure (see below for details). Fill the CPT         tubes completely and invert 5 times immediately after blood         collection.     -   2) Centrifugation. Check whether the CPT tubes fit into the         centrifuge bucket and that they do not touch the rotor in “swing         out” position. Gently invert the CPT tubes several times before         centrifugation. Centrifuge with 1650×g for 20 minutes at         18-25° C. in a horizontal centrifuge (swing out head), brake         “on”.     -   3) A white layer of PBMC should be visible above the stable gel         barrier. Gently invert the tube 3 times to suspend the cells in         the plasma. The gel barrier will stay intact.     -   4) Washing. Open the CPT tubes and transfer the cell suspension         from all 4 tubes into a 50 ml tube and fit up with washing         buffer. (Do not use washing buffers containing Calcium or         Magnesium). Spin down cells and resuspend in 1 ml PBS.     -   5) Wash paramagnetic particles. Transfer the paramagnetic         particles into a tube that fits in the magnet to be used for         separation. Add 1-2 ml PBS and put the tube into the magnet.         Wait for 1 minute (the solution should be clear) and take away         the PBS using a pipette without disturbing the particles. Add         0.5 ml PBS.     -   6) Add the resuspended cells to the paramagnetic particles and         incubate on ice or in +4° C. for 30 minutes with gentle tilting         (5-10 tilts/minute).     -   7) Add 3 ml PBS and isolate endothelial precursor cells by         putting the tube into the magnet for 2-3 minutes before         pipetting off the PBS and remaining cells. Do not throw away any         cells before you know that you have been successful in isolating         cells.     -   8) Wash the cells by resuspending them in PBS and repeating the         procedure above.     -   9) Divide the cells into at least three samples (positive         control, negative control and 1-2 unknown samples). Add 50 μl of         each serum to the cells with exception of the negative control         (positive control serum is not included in the kit). Mix and         incubate for 30 minutes in room temperature, mix gently after 15         minutes.     -   10) Wash the cells three times with cold PBS.     -   11) Add 10 μl secondary antibodies against IgM and IgG.     -   12) Incubate on ice or in +4° C. for 20 minutes.     -   13) Spin down the cells and wash with cold PBS before         resuspending in approximately 200 μl of PBS. Store samples on         ice or in +4° C.

14) Analyze samples by FACS, samples are defined as positive if the difference between the negative control and the sample is above 10 channels. TABLE 1 Antibodies used in immunocytochemical staining and flow cytometric analysis of Tie-2+ cells isolated from whole blood Immuno- Antibodies Company cytochemistry FACS CD1a Becton Dickinson − − (BD)-USA CD3 BD − − CD14 BD (+)/+ (+)/+ CD19 BD − − CD31 BD − − CD34 BD − − CD56 + 16 BD − − CD68 BD ++ ++ CD83 BD − − CD62E Biogenesis-UK +* +* (anti-E-selectin) CD106 Biogenesis-UK ++* ++* (anti-VCAM) CD54 R&D Systems-UK +* +* (anti-ICAM) VWF SEROTEC-UK ++ ++ Ac-LDL Molecular Probes, Inc-USA +++ +++ VEGF-R1 R&D Systems +++ +++ (Flt-1) MHC class I Serotec-UK +++ +++ MHC class II Serotec-UK + ++ MICA Dr. Thomas Spies (+)/+ (+)/+ Anti-Fibroblast Serotec-UK − − Anti-α-actin Boehringer Mannheim- − − Germany (+), weak or inconsistent staining; +, moderate staining; ++, strong staining; +++, very strong staining. vWF, vonWillebrand Factor; Ac-LDL, acetylated low-density lipoprotein; MICA, Major histocompatibility complex class I-related chain A. *expressed on Tie-2+ cells only after activation with TNF-alpha and IFN-gamma for 12-14 hrs.

TABLE 2 Flow cytometric analysis of reactivity of anti-endothelial-monocyte, endothelial specific and HLA class I or class II specific antibodies with a panel of Tie-2+ cells Pt. Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 No. Tie-2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Sera known to contain EM-reactive abs.  1 +/− +/− +/− +/− +/− +/−  2 +/− +/− +/− +/− +/− +/−  3 +/− +/− +/− +/− +/− +/−  4 +/− −/− −/− +/− −/− +/−  5 −/− +/− +/− +/− +/− −/−  6 −/− +/− +/− +/− +/− −/−  7 −/− +/− +/− +/− +/− −/−  8 −/− −/− −/− +/− −/− +/−  9 +/− +/− −/− −/− +/− −/− 10 +/− +/− −/− −/− +/− −/− Sera known to contain only EC-specific abs. 11 +/− +/− −/− +/− −/− −/− 12 +/− +/− −/− +/− −/− −/− 13 −/− −/− +/− −/− +/− +/− 14 −/− −/− +/− −/− +/− +/− 15 −/− −/− +/− −/− +/− +/− Sera known to contain only HLA class I broadly-reactive abs. (n = 5) 16 +/+ +/+ +/+ +/+ +/+ +/+ Sera known to contain only HLA class II abs. (n = 5) 17 +/+ −/− −/− +/+ −/− +/+ Control sera from pts. with stable graft function (n = 25) 18 −/−* −/− −/−* −/− −/− +/−* *3/25 (12%) sera reacted with some of the Tie-2 cells on the panel

TABLE 3 Reactivity of anti-endothelial-monocyte, endothelial specific and HLA class I or class II specific antibodies with a panel of Tie-2+ cells using the microcytotoxic assay Pt. Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 No. Tie-2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Tie2+/Tie2− Sera known to contain EM-reactive abs.  1 ++/− +++/− ++/− ++/− ++/− +++/−  2 ++/− +/− +++/− ++/− +++/− ++/−  3 +++/− ++/− ++/− ++/− ++/− ++/−  4 +++/− −/− −/− +++/− −/− +++/−  5 −/− ++/− ++/− ++/− ++/− −/−  6 −/− ++/− ++/− ++/− ++/− −/−  7 −/− ++/− ++/− ++/− ++/− −/−  8 −/− −/− −/− ++/− −/− ++/−  9 +++/− ++/− −/− −/− ++/− −/− 10 ++/− ++/− −/− −/− ++/− −/− Sera known to contain only EC-specific abs. 11 +++/− ++/− −/− +++/− −/− −/− 12 ++/− +++/− −/− ++/− −/− −/− 13 −/− −/− ++/− −/− ++/− ++/− 14 −/− −/− ++/− −/− ++/− ++/− 15 −/− −/− ++/− −/− ++/− ++/− Sera known to contain only HLA class I broadly-reactive abs. (n = 5) 16 +++/+++ +++/+++ ++++/++++ +++/+++ +++/+++ ++/++ Sera known to contain only HLA class II abs. (n = 5) 17 ++/++ −/− −/− ++/++ −/− ++/++ Control sera from pts. with stable graft function (n = 25) 18 −/−* −/− −/−* −/− −/− −/− *2/25 (8%) sera reacted with some of the Tie-2 cells on the panel −, negative; +, 10-25%; ++, 26-50%; +++, 51-75%; +++, 76-100% dead cells.

TABLE 4 Surface markers expressed by cells separated using paramagnetic particles coupled to antibodies specific for Tie-2. Surface molecule Stain (++ high or + low) HLA I ++ HLA II ++ CD31 ++ CD105 + CD151 ++ VEGFR-2 ++ CD133 + VEGFR-1 ++ CD68 ++ CD14 + CD62E + CD106 ++ CD54 + VWF ++ Ac-LDL ++

TABLE 5 Number of cells before Anti- Separation Number of cells separation × 10⁶ coagulant method after separation Yield (%) 78 EDTA Ficoll 0.35 0.4 62 EDTA Ficoll 0.3 0.5 64 ACD Ficoll 0.2 0.3 56 ACD Ficoll 0.3 0.6 85 ACD Ficoll 0.3 0.4 68 ACD Ficoll 0.5 0.8 54 ACD Ficoll 0.35 0.6 67 Heparin Ficoll 2.3 3.4 54 Citrate CPT tubes 1.0 1.9 52 Citrate CPT tubes 0.28 0.5 22 Heparin CPT tubes 1.0 4.5 36 Heparin CPT tubes 4.2 11.7 56 Heparin CPT tubes 2.1 3.8

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OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A kit for isolating an endothelial cell precursor cell in a sample comprising a first antibody that specifically binds an endothelial cell precursor cell surface marker and a detection reagent.
 2. The kit of claim 1, wherein said endothelial cell precursor cell surface marker is Tie2, HLA II, CD31, CD105, CD151, VEGFR-2, CD133, VEGFR-1, CD68, CD14, CD62E, CD106, CD54, VWF or Ac-LDL.
 3. The kit of claim 1, wherein the first antibody is a polyclonal antibody.
 4. The kit of claim 1, wherein the first antibody is a monoclonal.
 5. The kit of claim 1, wherein the kit further comprises written instructions for using the kit to determine whether a test sample contains an endothelial cell precursor cell.
 6. The kit according to claim 1, wherein the kit further comprises a positive control that comprises a polypeptide that comprises an antigenic determinant of an endothelial cell precursor cell surface marker protein, wherein the antigenic determinant binds to said first antibody.
 7. The kit according to claim 1, wherein the kit further comprises a negative control that comprises a polypeptide that does not comprises an antigenic determinant of an endothelial cell precursor cell surface marker protein.
 8. The kit of claim 1, wherein said sample is blood.
 9. The kit of claim 1, wherein said kit further comprises a sample collection means.
 10. The kit of claim 9, wherein said sample collection means further comprises an anti-coagulant.
 11. The kit of claim 10, wherein said anti-coagulant is heparin.
 12. The kit of claim 9, wherein said sample collection means is a CPT vacutainer tube.
 13. The kit of claim 1, wherein said detection reagent is a second antibody.
 14. The kit of claim 13, where said second antibody specifically binds human immunoglobin.
 15. The kit of claim 14, wherein said second antibody specifically binds an IgG or IgM immunoglobin.
 16. The kit of claim 13, wherein the second antibody is a polyclonal antibody.
 17. The kit of claim 13, wherein the second antibody is a monoclonal antibody.
 18. The kit of claim 1, wherein the first antibody is immobilized on a solid support.
 19. The kit of claim 18, wherein said solid support is, a particle, a microtiter plate, a cuvette, a tube, or a strip.
 20. The kit of claim 19, wherein said particle is a microparticle.
 21. The kit of claim 20, wherein said microparticle is a paramagnetic particle. 